Field of the Invention
The present invention relates to new hydrophilic copolymers, processes for their preparation and their use, in the form of aqueous gels, in separation techniques, such as gel permeation chromatography (also called gel filtration), and in techniques of immobilization of natural substances, especially proteins and in particular enzymes.
Gel permeation chromatography is a current method of fractionation and separation of molecules according to their size. Molecules of a size greater than the largest pores of the gel are displaced by the mobile liquid phase, although molecules of a smaller size penetrate more or less deeply into the pores of the gel, which constitutes the stationary phase, and are then more or less retained. If then a liquid phase, in which a sample containing molecules of different sizes has been injected, is made to circulate on a column filled with gel, the speed of migration of these molecules towards the bottom of the column will vary from one molecule to another and the molecules will then be separated.
The principal hydrogels used at present as supports in the techniques described above are on the one hand gels of natural polysaccharides possibly cross-linked (agarose gels, dextran gels known under the trademark Sephadex) and, on the other hand, gels of synthetic or semisynthetic polymers such as, for example, the gels of polyacrylamide or the polyacrylamide-agarose mixed gels. These supports are not free of defects. The gels of polyacrylamide or polyacrylamide-agarose are chemically unstable in a basic medium owing to hydrolysis of the amide groups into carboxylic acid groups, which limits their field of use. Certain gels of dextran have moderate mechanical properties, and hence it is impossible to make columns thereof having a relatively high and constant rate of flow. Finally, the agarose gels have a poor resistance to heat and to agents for weakening the hydrogen linkages (urea, guanidine, etc.), and further, like all gels of natural origin, are very sensitive to attack by bacteria or certain enzymes.
Because of the defects described above, it has been proposed to replace the gels set forth above by gels of hydrophilic copolymers of N-[tris(hydroxymethyl)-methyl]methacrylamide and of N,N'-ethylene-bis-methacrylamide or N,N'-methylene-bis-acrylamide (cross-linking agent) (cf. Tetrahedron Letters No. 6, pgs. 357-358, 1975). However, to obtain homogeneous gels, therefore transparent, of such copolymers is difficult since, during the polymerization, there is formed, especially in the case of high concentrations of cross-linking agent, homopolymers of N,N'-ethylene-bis-methacrylamide or N,N'-methylene-bis-acrylamide, which form white precipitates in the gel.